Protective impact of Spirulina platensis against γ-irradiation and thioacetamide-induced nephrotoxicity in rats mediated by regulation of micro-RNA 1 and micro-RNA 146a

Abstract

Chronic kidney disease develops popular and medical health problems, especially in developing countries. The objective of this study is to investigate the protective mechanism of Spirulina platensis against γ-irradiation (R) and/or thioacetamide (TAA)-induced nephrotoxicity in rats. Rats intoxicated with R or TAA showed alterations in kidney function markers (urea, creatinine, albumin, and total protein contents), oxidative stress markers (malondialdehyde, reduced glutathione), antioxidant enzymes (superoxide dismutase, catalase), and several inflammatory markers (including, the high-sensitivity C-reactive protein, hypoxia-inducible factor-1 alpha, tumor necrosis factor-alpha, interferon-gamma, some interleukins, and nuclear factor-kappa B). Rats also acquired apoptosis, evinced by high caspase-3 efficacy. This nephrotoxicity mediated by upregulation of the messenger RNA (mRNA) gene expression of the autophagy markers: Beclin-1, microtubule-associated protein LC3, p62 binding protein, immunoglobulin G receptor Fcγ receptor (FcγR), micro-RNA-1 (miR-1), protein expression of phospho-adenosine monophosphate-activated protein kinase, and phospho-mammalian target of rapamycin, along with downregulation of miR-146a mRNA gene expression and alteration of calcium and iron levels. The combined treatment R/TAA enhanced the observed oxidative stress, inflammation, apoptosis, and autophagy that mediated by higher upregulation of miR-1 and downregulation of miR-146a mRNA gene expression. Spirulina platensis administration exhibited a nephroprotective impact on R, TAA, and R/TAA toxicities via regulating miR-1 and miR-146a mRNA gene expression that monitored adenosine monophosphate-activated protein kinase/mammalian target of rapamycin signaling.

Graphical Abstract

Introduction

Chronic kidney disease (CKD) is a global health problem with high morbidity and mortality rates. CKD is closely correlated with diabetes and cardiovascular diseases, which enhances the popular and medical health problems especially in the developing countries [1, 2]. Prolonged exposure to environmental pollutants as well as drug administration are the main cause of CKD. During the progress of CKD, the kidneys suffers from continuous oxidative stress, inflammation, mitochondrial damage, and apoptosis. Moreover, CKD may end with kidney fibrosis and renal failure. The gradual increase of kidney impairment during CKD leads to the accumulation of wastes, chemicals, electrolytes, and fluids within different organs that results in tissues injury [3–5].

A significant source of exogenous as well as endogenous reactive oxygen species (ROS) production in the body is ionizing radiation. The subjection to ionizing radiation takes place due to the naturally occurring radioactive contaminants in the atmosphere or, particularly, during radiotherapy [6]. Radiotherapy is commonly used to fight cancer. The exposure to γ-irradiation (R) may result in the induction of oxidative stress and destructive effects of the radiosensitive organs, including the kidneys, i.e. inducing renal nephropathy. The released ROS during exposure to R can cause several renal toxic reactions, which results in renal cell disturbance, alteration in the membrane structures and trace elements’ levels, as well as the destruction of macromolecules (DNA and RNA). These events end with cell death [7–9].

Thioacetamide (TAA) is a water-soluble, sulfur-containing organic compound, with the molecular formula C₂H₅NS (Supplementary Fig. 1), is a source of sulfide ions in the organic and inorganic chemical synthesis. TAA is used as a fungicide in agriculture as well as, in several other industries, demonstrates several toxicological properties [10–13]. TAA toxicity is mediated by oxygenations of its thioamide-sulfur atom, catalyzed by flavoprotein monooxygenases and/or cytochrome P450, followed by the formation of reactive thioacetamide-S-oxide metabolites and production of ROS [13]. Although the liver is the primary target organ for TAA toxicity, it may extend to other organs, including the kidneys [13, 14]. Numerous cellular and biochemical changes accompanied by alterations of the trace elements’ contents have been categorized in TAA-intoxicated kidneys [14–17].

Spirulina platensis (SP) is a cyanobacterial microalgae, which contains high-functioning components, including proteins, polyunsaturated fatty acids, accompanied by several bioactive compounds, such as C-phycocyanin, allophycocyanin, vitamins, β-carotene, and minerals that give rise to anti-inflammatory and antioxidant properties [18–22]. Accordingly, it is used as a safe natural dietary supplement for humans and as a feed additive for animals [22–24]. SP exhibits scientific importance because of its broad range of biological actions, including anticancer [25], hepato-, and renal-protective activities [20, 21, 25–28]. SP protective activity is mainly due to its C-phycocyanin constituents [19–21, 27, 28].

Accordingly, the objective of this study is to investigate the protective mechanism of SP against R- and/or TAA-provoked nephrotoxicity in rats.

Materials and Methods

Animals

Wistar male albino rats (weighing 180–200 g) were obtained from the breeding unit of the National Center for Radiation Research and Technology (NCRRT). The animals were held under standard sanitary conditions—at temperature (23 ± 4°C), humidity (65 + 5%), with 12-h/12-h light/dark sequences illumination conditions. They were freely allowed to standard pellet diet (21% protein) and freshwater ad libitum. Animals, at the NCRRT animal house, were acclimatized 1 week in laboratory environment prior to the onset of the experiment. The international guidelines for animal experiments were performed. The Ethical Committee of the NCRRT approved this study (No. 15A/19).

Irradiation facilities

The animals, in groups of 10, were exposed to whole-body γ-irradiation, at NCRRT, using Canadian Gamma Cell-40 (¹³⁷Cs, manufactured by the Atomic Energy of Canada Limited, Ontario, Canada) that belongs to the Egyptian Atomic Energy Authority. The radiation dose rate was 0.403 Gy/min. The total dose of radiation was 8 Gy (1 Gy fractional dose twice per week for 4 weeks of (1 Gy × 2 × 4) experimental period.

Experimental design

The rats were distributed randomly into 8 groups of 10 (the sample size computation represented in Supplementary data) for the 4-week experimental period. Group 1, (C): rats held as control; Group 2, (SP): rats received 500 mg/kg body weight (b.w.) SP extract (imported from Malaysia by Imtenane herbal drug store, Nasr city, Cairo, Egypt), dissolved in distilled water, daily via gastric gavage for 4 weeks [27]; Group 3, (R): rats exposed to γ-radiation at a dose of 1 Gy twice per week for 4 weeks; Group 4, (TAA): rats injected with 150 mg of TAA (Sigma-Aldrich Co., St. Louis, MO)/kg b.w. intraperitoneally (IP) twice per week for 4 weeks [17]; Group 5, (R/TAA): rats exposed to 1 Gy of γ-radiation, a day before administration of 150 mg TAA/kg b.w., IP, twice per week for 4 weeks; Group 6 (SP/R): rats, which received 500 mg/kg b.w. of SP extract orally every day for 4 weeks, were exposed to γ-radiation twice per week, Group 7 (SP/TAA): rats, which received 500 mg/kg b.w. of SP orally every day, administered 150 mg TAA/kg b.w., IP, twice per week for 4 weeks, Group 8 (SP/R/TAA): rats, which received 500 mg/kg b.w. of SP orally every day, were exposed to 1 Gy of γ-radiation a day before administration of 150 mg TAA/kg b.w., IP, twice per week for 4 weeks.

Twenty-four hours after the last treatments, all the animals were anesthetized with 1.2 g/kg b.w. urethane (Sigma-Aldrich) [29]. Cardiac blood samples were collected in ethylenediamine tetraacetic acid (EDTA) tubes for hematologic research and in anticoagulant-free glass tubes held to coagulate and then centrifuged to separate the blood serum for biochemical examination. The kidneys from each rat were immediately excised, rinsed in ice-cold ionized water, dried carefully, weighed, and stored at −80°C for subsequent analyses.

For histopathological investigations, kidney tissue samples were fixed in a 10% neutral buffered formalin solution. Tissue specimens were dehydrated in ascending ethanol concentration, washed off in xylene, embedded in paraffin wax, and sliced at a 5-μm thickness. The obtained slides’ sections were stained by hematoxylin and eosin (H&E) and then examined under electric microscope [30]. The following scores were applied for the grading system for renal lesions, 0: normal histology; 1: tubular epithelial cell degeneration, without significant necrosis or apoptosis; 2: tubular epithelial cell necrosis and apoptosis < 25%, with tubular epithelial cell necrosis and apoptosis < 50%; 3: tubular epithelial cell necrosis and apoptosis < 25%; 4: tubular epithelial cell necrosis and apoptosis < 75%; 5: tubular epithelial cell necrosis and apoptosis ≥75% [31].

Biochemical assessments

Blood biochemical parameters: urea, creatinine, albumin (ALB), and total proteins (TP) were measured in serum using VITROS 350 Reference Fluid, Micro Slide Assay (Dry Chemistry System, Ortho-Clinical Diagnostics, Inc., Johnson & Johnson, Linden, NJ) at the Biological Fluids Lab, Regional Center for Food and Feed, Agriculture Research Center, Giza, Egypt.

Hematologic indices

The automated Hematology-Analyzer (XT-2000i, Sysmex Corporation, KOBE, Japan) at the Biological Fluids Lab, Regional Center for Food and Feed, Agriculture Research Center, Giza, Egypt, was used to determine the red blood cells (RBCs) count, hemoglobin (HB) concentration, and hematocrit percentage (HCT%) in EDTA blood samples.

Preparation of crude kidney’ homogenate

One gram of the kidney tissues was homogenized in ice-cold potassium phosphate-buffered saline (50 mM, pH 7.4) [16] using a Teflon homogenizer centrifuged at 1200 g for 15 min at 4°C to prepare a 10% (w/v) kidney homogenates, which is used in the following biochemical assessments.

Special biochemical parameters

Lactate dehydrogenase (LDH), sodium (Na⁺), potassium (K⁺), and chlorine (Cl⁻) in the kidney tissues were assessed using commercial kits (DiaSys Diagnostic Systems GmbH, Germany). Ferritin, in the kidney tissues, was assessed using Ferritin-Rat-ELISA Kit (MBS564109, MyBioSource).

Determining oxidative stress parameters and antioxidant enzymes

Biodiagnostics kits were used to determine superoxide dismutase (SOD) [32] and catalase (CAT) [33] activities as well as for the determination of the malondialdehyde (MDA) [34] level and the reduced glutathione (GSH) [35] contents in the kidney tissues.

Determining the inflammatory and apoptotic markers

MyBioSource commercial ELISA kits were used for assessment of the high-sensitive C-reactive protein (hs-CRP, Cat.No:MBS008334), hypoxia-inducible factor-1 alpha (HIF-1α, Cat.No:MBS028091), interleukin-1beta (IL-1β, Cat.No:MBS825017), interleukin-6 (IL-6, Cat.No:MBS355410), interleukin-17 (IL-17, Cat.No:MBS164772), tumor necrosis factor-alpha (TNF-α, Cat.No:MBS824824), interferon-gamma (IFN-γ, Cat.No:MBS267008), and nuclear factor-kappa B (NF-κB, Cat.No:MBS268833) levels in the kidney tissues.

The activity of the apoptotic marker caspase-3 estimated by R&D Colorimetric Assay Kit, (Cat.No:BF3100) following the experimental guidelines. The caspase-3 activity calculated as ng/ml.

Real-time quantitative reverse transcription-polymerase chain reaction

Qiagen kit (USA) was used to extract the total RNA from the frozen kidney pieces, which then inversely transcripted into complementary DNA (cDNA), employing Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Madison, USA). Step One Plus Real-Time polymerase chain reaction (PCR) System (Applied Biosystems, Foster City, CA) and an SYBR® Green PCR Master Mix (Applied Biosystems) were conducted in a 10-μl final volume, programming the heating cycles: 95°C (10 min), then 40 cycles of 95°C (15 s) and 65°C (1 min). The sequences of PCR primer pairs and the housekeeping reference gene beta-actin (β-actin) with the corresponding bank gene accession number are denoted (Supplementary Table 1). The data were evaluated with the ABI Prism sequence detection system software and computed using v1.7 Sequence Detection Software from PE Biosystems (Foster City, CA). The relative expression values of the studying genes were evaluated using the comparative threshold cycle method. All values were normalized to β-actin, applying the expression (2^−ΔΔCt) [36].

Micro-RNAs expression

MirVana™ micro-RNA (miRNA) isolation Kit (Life Technologies Corporation, USA, Cat.No:AM1560) was utilized to extract the total miRNA from kidney tissues. The extracted miRNA was reverse transcribed to cDNA using TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, USA, Cat.No:4366597). The quantification of miRNA from cDNA was accomplished with the primer of the miRNA-1 (rno-miR-1, MyBioSource, MBS825354, gene bank accession: NC_005117.4) and the miRNA-146a (rno-miR-146a, MBS825223, gene bank accession: NC_005109.4). These kits contain the SYBR Green dye. Each miRNA relative expression level was calculated as 2^−ΔΔCt after normalization to the expression of the housekeeping gene RNU6 in each examined sample [36].

Western blot analysis

TRIzol reagent was used to extract tissue proteins and then Lowry method was utilized to estimate the TP concentrations [37]. Twenty micrograms of protein per lane were isolated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transported to polyvinylidene fluoride membranes. Membranes were then incubated at room temperature for 2 h with a blocking solution (5% nonfat dried milk/10 mM Tris–HCl/pH 7.5/100 mM NaCl/0.1% Tween 20). Membranes were incubated overnight at 4°C with the designated primary antibodies phospho-adenosine monophosphate-activated protein kinase (p-AMPK, 1:300, Cat.No: PA5-104982), phosphorylated mammalian target of rapamycin (p-mTOR) (Ser2454) polyclonal antibody (Cat.No:PAS5-105571, Invitrogen), and β-actin (1:500, Cat.No:MA5-1140, Invitrogen, Thermo Fisher Scientific), then incubated with a mouse anti-rabbit secondary monoclonal antibody coupled to horseradish peroxidase at room temperature for 2 h. Thereafter, the membranes were washed several times (with 10 mM Tris–HCl/pH 7.5/100 mM NaCl/0.1% Tween 20) after each incubation at room temperature. Chemiluminescence detection was achieved with the Amersham detection kit. The amount of the analyzed protein was computed by densitometric analysis using BioRad software, USA. The results were normalized to β-actin protein expression. The protein level was expressed relative to β-actin.

Determining the calcium and iron levels

The kidney tissues of different studied groups were digested in a mixture of concentrated nitric acid (HNO₃) and hydrogen peroxide (H₂O₂) (5:1 v/v) until over all digestion of organic materials using Milestone MLS-1200 Mega, High-Performance Microwave Digester Unit, Italy. The calcium (Ca²⁺) and iron (Fe²⁺) levels were estimated in prepared tissue samples using an Atomic Absorption spectrophotometer (Thermo Scientific, iCE 3000, UK) at the NCRRT.

Statistical analysis

The Statistical Package for Social Science Software program (version 21.0) and Microsoft Excel were used to analyze the data. The data were explained as the mean ± standard error. One-way analysis of variance with the least significant difference post hoc multiple comparisons were used to test the variation in the means of the variables among different groups. The probability of P < 0.05 was thought to be significant.

Results

The data demonstrated that R and TAA achieved synergistically in a dose–response manner to induce nephrotoxicity.

Biochemical Parameters

Exposure to R, TAA injections, and R/TAA treatments resulted in significant increases of urea (2, 2.5, and 4.1-folds) and creatinine (2.6, 2.8, and 4.6-folds) levels (P < 0.01) along with a significant (P < 0.01) decline in serum ALB (72.2%, 63.6%, and 54.4%) and TP (69.0%, 66.3%, and 58.3%), respectively, as compared with the corresponding controls. However, SP/R treatment animals exhibited significant declines (P < 0.05) in the urea and creatinine levels, with significant increases in ALB and TP contents compared with R group. Similarly, SP/TAA-treated animals demonstrated significant decreases (P < 0.05) in the urea and creatinine levels, with significant increases in ALB and TP contents compared with TAA-intoxicated group. SP/R/TAA combined treatment displayed significant (P < 0.05) amelioration in the urea, creatinine, ALB, and TP contents compared with R/TAA-intoxicated animals (Fig. 1).

Figure 1.

Biochemical Parameters. C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05). ALB: albumin, TP: total protein.

Hematologic indices

RBCs count (59.5%, 59.3%, and 51.6%), HB concentration (72.3%, 69.8%, and 63.0%), and HCT% (73.7%, 70.7%, and 65.5%) declined in the R, TAA, and R/TAA-intoxicated groups. However, SP treatment verified significant changes (P < 0.05) of these hematologic indices compared with control, R, TAA, and R/TAA groups. SP treatment preserved RBCs count (89.5%, 90.9%, and 69.5%), HB concentration (90.6%, 90.8%, and 83.9%), and HCT% (93.5%, 88.6%, and 80.2%) in the R, TAA, and R/TAA-intoxicated groups, respectively, compared with control values. Conversely, SP/R, SP/TAA, and SP/R/TAA groups revealed considerable enhancement (P < 0.05) in the RBCs count, HB concentration, and HCT% compared with R, TAA, and R/TAA-intoxicated groups, respectively (Fig. 2).

Figure 2.

Hematologic indices. C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05). RBCs: Red Blood Cells, HB: hemoglobin, HCT: hematocrit, mc/μL: million cells per microliter, g/dL: gram per deciliter.

Sodium, potassium, and chloride levels

Kidney tissues of R, TAA, and R/TAA-intoxicated animals showed suggestive increase (P < 0.05) of both Na⁺ (1.3, 1.3, and 1.5-folds) and Cl⁻ (1.3, 1.5 and 1.7.0-folds) levels accompanied by a significant decline (P < 0.05) in the K⁺ level (79.7%, 74.2%, 65%), respectively, compared with the controls. On the contrary, SP/R, SP/TAA, and SP/R/TAA groups revealed considerable declines (P < 0.05) in the renal Na⁺ and Cl levels accompanied with nonsignificant variations (P > 0.05) of renal K⁺ levels compared with R, TAA, and R/TAA groups, respectively (Fig. 3).

Figure 3.

Elements and Ferritin levels in Kidney tissues. C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05). Na: sodium, K: potassium, Cl: chloride, Ca: calcium, Fe: iron.

Trace elements

Kidney tissues of R, TAA, and R/TAA-intoxicated animals showed a significant (P < 0.05) increase of Ca²⁺ level (2.4, 2.5, and 3.7-folds) accompanied by a significant decline of the Fe²⁺ level (69.4%, 65.7%, and 48.5%) compared with the controls. In contrast, SP/R, SP/TAA, and SP/R/TAA groups considerably (P < 0.05) restored the renal Ca²⁺ and Fe²⁺ levels in the kidney tissues compared with R, TAA, and R/TAA groups, respectively (Fig. 3).

Ferritin

A significant (P < 0.05) decline in the ferritin level (61.7%, 57.4%, and 46.1%) was observed in the kidney tissues of R, TAA, and R/TAA-intoxicated animals compared with the controls. SP treatment significantly ameliorated (P < 0.05) the ferritin level in the R and R/TAA-intoxicated groups (Fig. 3).

Antioxidant enzymes

Kidney tissues of R, TAA, and R/TAA-intoxicated animals showed considerable (P < 0.05) inhibitions of SOD (57.8%, 48.8%, and 40.6%) and CAT activities (55.2%, 52.5, and 36.7%) compared with the controls. Adversely, SP treatment significantly (P < 0.05) improved SOD and CAT activities in the R, TAA, and R/TAA-intoxicated groups (Fig. 4).

Figure 4.

Superoxide dismutase (SOD) and Catalase (CAT) activity, malondialdehyde (MDA) level and glutathione (GSH) content in the kidney tissues. C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05).

MDA and GSH

Kidney tissues of R, TAA, and R/TAA-intoxicated animals revealed a suggestive (P < 0.05) enhancement of MDA level (1.5, 1.7, and 3.0-folds), whereas GSH content considerably (P < 0.05) declined (68.2%, 64.4%, and 34.3%) in the kidney tissues of R, TAA, and R/TAA-intoxicated animals, respectively, compared with their controls. SP treatment significantly amended the MDA and GSH contents in the kidney tissues of R, TAA, and R/TAA-intoxicated animals, respectively (Fig. 4).

LDH activity

Exposure to R, TAA injections, and their combined toxic effect (R/TAA) resulted in a significant (P < 0.05) increase of kidney LDH activity (1.7, 1.9, and 2.1-folds), respectively, compared with the control values. Even so, SP/R, SP/TAA, and SP/R/TAA treatments exhibited significant decreases (P > 0.05) of LDH activity compared with R, TAA, and R/TAA groups, respectively (Fig. 5).

Figure 5.

Lactate Dehydrogenase activity, High-Sensitivity C-Reactive Protein, Hypoxia Inducible Factor-1 alpha and the inflammatory markers’ levels in kidney tissues. C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05). IL-1β: Interleukin-1 beta, IL-6: Interleukin 6, IL-17: Interleukin 17, TNF-α: Tumor Necrosis Factor-alpha, INF-: Interferon-gamma, NF-κB: Nuclear Factor-kappa B.

The hs-CRP and HIF-1α

Kidney tissues of R, TAA, and R/TAA-intoxicated groups showed a significant (P < 0.05) enhancement of the renal levels of both hs-CRP (1.8, 2.3, 3.5-folds) and HIF-1α (4.3, 5.1, 7.2-folds) compared with the control values. However, SP treatment significantly (P > 0.05) depressed the hs-CRP and HIF-1α renal levels in the R, TAA, and R/TAA groups (Fig. 5).

The pro-inflammatory markers

Kidney tissues of R, TAA, and R/TAA-intoxicated groups showed considerable increases of IL-1β (3.0, 5.3, and 7.0-folds), IL-6 (2.6, 2.7, and 3.6-folds), IL-17 (4.4, 4.8, and 5.6-folds), TNF-α (3.1, 3.3, and 4.3-folds), IFN-γ (4.9, 6.5, and 8-folds), and NF-κB (2.8, 3.4, and 4.3-folds) levels compared with the control levels. However, SP treatment significantly downregulated the renal levels of these inflammatory marker in the R, TAA, and R/TAA-intoxicated groups (Fig. 5).

Apoptosis

Kidney tissues of R, TAA, and R/TAA-intoxicated groups showed considerable elevated levels of caspase-3 (5.6, 6.2, and 11.0-folds) compared with the control levels. On the other hand, SP treatment displayed significant downward level (P > 0.05) of the renal caspase-3 activities in the R, TAA, and R/TAA-intoxicated groups (Fig. 6).

Figure 6.

Caspas-3 activity in kidney tissues. C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05).

Autophagy

Kidney tissues of R, TAA, and R/TAA-intoxicated groups demonstrated significant (P < 0.05) downregulations of the messenger RNA (mRNA) gene expression of Beclin-1 (54.4%, 38.7%, and 21.2%), microtubule-associated proteins 1 light chain 3 (MAP1 LC3; LC3) (49.8%, 38.8%, and 19.6%) accompanied by upregulation of immunoglobulin G (IgG) receptor [Fcγ receptor (FcγR)] (4.1, 4.8, 7.4-folds), and ubiquitin-binding protein p62 mRNA (1.9, 3.0, and 6.4-folds) compared with the corresponding controls. SP/R, SP/TAA, and SP/R/TAA treatments significantly (P > 0.05) ameliorated the renal LC3, FcγR, and p62 mRNA gene expressions compared with R, TAA, and R/TAA groups, respectively (Fig. 7).

Figure 7.

Beclin 1, Microtubule-associated proteins 1 light chain 3 beta (MAP1 LC3β; LC3), Ubiquitin-binding protein  p62 mRNA, IgG receptor (FcγR), micro-RNA − 1 (miR-1) and micro-RNA -146a (miR-146a) mRNA gene expressions. C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05).

Micro-RNA

A suggestive (P < 0.05) upregulation of the miR-1 gene expression (6.0, 7.7, and 10.3-folds) and a downregulation of the miR-146a gene expression (67.1%, 39.3%, and 23.6%) was recorded in the R, TAA, and R/TAA-treated groups, respectively, compared to the controls. SP/R, SP/TAA and SP/R/TAA treatments significantly (P > 0.05) remodeled the miR-1 and miR-146a gene expressions, compared with R, TAA, and R/TAA-intoxicated groups, respectively (Fig. 7).

p-AMPK and p-mTOR protein expression

Kidney-tissues of R, TAA, and R/TAA-intoxicated groups demonstrated significant (P < 0.05) upregulations of p-AMPK protein expression ratios (4.9, 5.1, and 7.4-folds) and p-mTOR protein expression ratios (3.2, 3.7, and 5.5-folds) compared with the corresponding controls. However, SP administration showed a significant (P < 0.05) promotion of p-AMPK protein expression ratios and downregulated p-mTOR protein expression ratios in the kidney tissues of R, TAA, and R/TAA-intoxicated animals (Fig. 8).

Figure 8.

Phoshpho-Adenosine monophosphate-activated protein kinase (p-AMPK) and phospho-mammalian target of rapamycin (p-mTOR) protein expressions, relative to Beta actin (β-Actin). C: control, SP: Spirulina platensis treated animals, R: gamma-irradiated animals, TAA: Thioacetamide treated animals, R/TAA: gamma-irradiation/ thioacetamide treated animals, SP/R: Spirulina platensis/gamma-irradiation treated animals, SP/TAA: Spirulina platensis/Thioacetamide treated animals, SP/R/TAA Spirulina platensis/gamma-irradiation/Thioacetamide treated animals. Significance to control (^a), significance to R (^b), significance to TAA (^c), significance to R/TAA (^d) at p < .05).

Histopathology

The histopathological investigations of kidney sections (H&E ×400) were demonstrated in Fig. 9. The C and SP kidney tissue sectors displayed a typical histological structure characterized by circumscribing glomeruli with normal of capillary tufts and Bowman’s capsule structure (Fig. 9a and b). The renal tubules of both proximal and distal convoluted tubules showed intact epithelial lining and regular arrangement (score 0). However, R, TAA, and R/TAA-intoxicated animals exhibited kidney tissues structural alterations. Kidney tissue of R-group revealed mild histological changes of the renal tubular epithelial lining, which appeared in the form of swelling of cell lining with intraluminal albuminous casts (Fig. 9c). Tubular epithelial cells showed degeneration without significant necrosis or apoptosis. Kidney tissue section showed severe congestion of glomerular tufts and interstitial blood capillaries (score 1). The kidney tissue of the TAA-treated rats revealed shrinkage of capillary tufts with widening of Bowman’s space of some glomeruli. The renal tubules showed epithelial cell degeneration with marked swelling of tubular epithelial lining accompanied by narrowing and occlusion of tubular lumen by albuminous and cellular casts. Tubular epithelial cell necrosis and apoptosis <25% (score 2) were seen. Interstitial edema and a few mononuclear cells infiltration mainly lymphocytes and macrophages were also noticed (Fig. 9d). However, kidney tissue of R/TAA-intoxicated animals revealed shrinkage of capillary tufts with widening of Bowman’s space of some glomeruli and degeneration of the renal tubular epithelial lining appeared in the form of swelling and granularity of its cytoplasm. Intratubular albuminous eosinophilic casts and congestions of interstitial blood capillaries were observed. Tubular epithelial cell necrosis and apoptosis <75% was seen (score 4) (Fig. 9e). In contrast, SP administration protected kidney tissues and minimized the observed structural alterations in the kidney tissues of intoxicated groups. The kidney tissue of the SP/R group revealed mild histological changes of the renal tubular epithelial lining; this appeared in the form of swelling of the tubular epithelial lining without significant necrosis or apoptosis. The glomeruli showed a mild degree of shrinkage of the glomerular tufts (score 1) (Fig. 9f). The kidney tissue of SP/TAA animal group showed shrinkage of capillary tufts accompanied with widening of Bowman’s space of some of these glomeruli. The renal tubules showed epithelial cell degeneration with marked swelling of the tubular epithelial lining accompanied by intratubular albuminous droplets. Tubular epithelial cell necrosis and apoptosis were seen (score 1) (Fig. 9g). The kidney tissue of SP/R/TAA-treated animals showed shrinkage of capillary tufts with widening of Bowman’s space of some glomeruli and degeneration of renal tubular epithelial lining appeared in the form of swelling and granularity of its cytoplasm. Intratubular albuminous eosinophilic casts and tubular epithelial cell necrosis and apoptosis <25% (score 2) were also observed (Fig. 9h).

Figure 9.

Kidney histopathological investigation. Photomicrograph of the kidney tissue section showing the normal structure; glomeruli and renal tubules of the C (control, 9a) and SP (Spirulina platensis treated animals, 9b), severe congestion and dilatation of capillary tufts in R (gamma-irradiated animals, 9c), shrinkage of capillary tufts in TAA (Thioacetamide treated animals, 9d), interstitial edema and few mononuclear cells infiltration in R/TAA (gamma-irradiation/thioacetamide treated animals, 9e), swelling and granularity of tubular epithelial lining and congestion of interstitial capillaries, intact and regular arrangement of tubular epithelial lining of SP/R (Spirulina platensis/gamma-irradiation treated animals, 9f), swelling of tubular epithelial lining and congestion of glomerular tufts in SP/TAA (Spirulina platensis/Thioacetamide treated animals, 9 g), mild swelling of tubular epithelial lining and hyper-cellular of capillary tufts in SP/R/TAA (Spirulina platensis/gamma-irradiated/Thioacetamide treated animals, 9 h) (H&E x400).

Discussion

Significant changes in kidney markers (urea, creatinine, ALB, and TP) in the serum as well as alterations of LDH activity, Na, K, Cl, Ca²⁺, and Fe²⁺ levels in the kidney tissues were distinguished in the R, TAA, and R/TAA-treated rats. Former studies indicated a physiological disturbance in kidney function parameters due to R exposure [7–9, 38, 39] or TAA administration [16, 17]. R exposure engenders amino acid oxidative deamination, protein catabolism, alterations of the membranes’ permeability, and damage of the tubular epithelium and other tubulointerstitial components, which impairs kidney function and provokes electrolyte imbalance [40]. The increased levels of urea and creatinine imply insufficient renal function. Animal studies have established that tubular injury plays a central role in the reduction of glomerular filtration rate in acute tubular necrosis. Obstruction and back leak of glomerular filtrate could be involved in the glomerular dysfunction in TAA-treated rats, which may be observed secondary to ROS release. TAA also induced mesangial cell contraction, altering filtration surface area, and modifying the ultrafiltration coefficient factors that decrease glomerular filtration rate [15, 41]. However, SP administration preserved the kidney microscopic alterations in the intoxicated animals and protected the kidney of different nephropathy-induced animal [5, 42].

The hematologic indices RBCs count, HB concentration, and HCT% declined in R, TAA, and R/TAA-intoxicated groups. These results coincide with those of previous research [40, 41, 43, 44]. On the other hand, ferritin and iron concentrations were diminished in the kidney tissues. R-exposure [45, 46] and TAA treatment [47] breakdown the hematopoietic system and decrease the iron level in the kidney and blood. Iron is an essential element that regulates erythropoiesis in bone marrow. The decline of iron level can induce anemia and hematopoietic system failure [48, 49]. Low concentration of ferritin reflects little stored iron, indicating an exhausted iron pool in the kidney [48]. Low concentrations of iron results in decline levels of ferritin synthesis, and vice versa [50]. These results indicate that R and TAA induced oxidative stress, which interrupts the iron metabolism, induced iron deficiency anemia, and depleted iron and ferritin levels in the kidney tissues. However, SP administration recovered the hematopoietic system via enhancing RBCs count, HB concentration, HCT%, iron, and ferritin concentrations. SP demonstrates similar recovery of the hematopoietic system in furan-intoxicated rats [49].

As mentioned above, R and TAA-induced oxidative stress is confirmed in the present study via MDA elevated levels, GSH consumption, and inhibition of SOD and CAT activities. Previous studies also confirmed these results [ 4, 7, 9, 51]. As mentioned above, TAA toxicity is mediated by oxygenations of its thioamide-sulfur atom catalyzed by flavoprotein monooxygenases and/or cytochrome P450, followed by the formation of reactive thioacetamide-S-oxide metabolites and production of ROS [13]. ROS can induce lipid peroxides overproduction and GSH depletion during the antioxidant defense mechanism, which could develop apoptosis and necrosis in kidney tissues. SP administration was observed to improve the antioxidant status in kidney tissues via amelioration of MDA level, GSH content, and activation of SOD and CAT. SP treatment also enhanced the antioxidant status in kidney tissues against cyclophosphamide and nicotine toxicities in rats [27, 28].

The data of the recent investigation demonstrated statistical elevations in the inflammatory markers’ levels: IL-1β, IL-6, IL-17, TNF-α, IFN-γ, and NF-κB due to R-exposure, TAA-administration, or R/TAA treatments. R-exposure triggered an inflammatory response, as described previously [9]. R-induced upregulations of the gene expression ratios of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and an increase in NF-κB protein expression in the rats’ kidneys [27]. Also, Zargar et al. [52] reported an increase of TNF-α, IL-4, and IFN-γ levels in kidneys of TAA-intoxicated rats. Synergistic correlation is also observed between the pro-inflammatory cytokines and NF-κB to induce renal inflammation. Pro-inflammatory cytokines are associated with the activation of the NF-κB, at the same time, NF-kB triggers the release of pro-inflammatory cytokines [11, 52]. In the current study, SP administration showed opposing results, as it regulated the levels of the studied pro-inflammatory markers. SP treatment also regulated the level of pro-inflammatory cytokines (IL-6 and TNF-α) and the protein expression ratio of NF-κB in the kidneys of nicotine-intoxicated rats [28]. SP demonstrated nephroprotection against the released pro-inflammatory cytokines-induced renal toxicity via driving down the oxidative stress and inhibiting the NF-κB signaling pathway.

The results also illustrated elevation of the apoptotic marker; caspase-3 levels in the kidneys of R-, TAA- and R/TAA-intoxicated groups. These results validate those of Soliman et al. [7] and Keshk and Zahran [4], who recorded an upregulation of caspase-3 protein expression in the kidney of irradiated and TAA-intoxicated rats. Oxidative stress and inflammation cooperate to induce apoptosis or programmed cell death. Apoptosis is a highly prevalent destructive effect of R-exposure [7]. SP administration can inhibit the release of the pro-inflammatory cytokines via blocking apoptosis [53]. SP administration contradicted the observed apoptosis by regulating the apoptotic pathway, terminated with decay of the caspase-3 level [28, 53], which is confirmed by the present work.

Moreover, autophagy, a relevant response to oxidative stress for managing cellular stability, correlates with different kidney disorders [54, 55]. Significant autophagy biomarkers: Beclin-1 and LC3 participate in autophagic programmed cell death [54–57]. Beclin-1 takes a part in controlling autophagosomes consistency. Beclin-1 initiates autophagy, whereas it is connected with Bcl2. Death-associated protein kinase 1 then phosphorylates and disconnects the Beclin-1/Bcl-2 complex, consequently stimulating the autophagy route [56–59]. Autophagosome formation demands LC3 MAP, an autophagy biomarker. The cysteine protease Atg4 splits pre-LC3 to LC3A, at the cytoplasm. Atg7, another cysteine protease, then, activates LC3A to its LC3B membrane-bound form situated in the autophagosomes’ inner and outer membranes. LC3B cooperates in the prolongation of the membrane; in contrast, alteration of LC3 coordinates with autophagic activation and renal damage acceleration [54, 56, 58, 60]. The autophagy receptor p62 has a reliable task during oxidative stress via attaching to autophagy substrates; it is a nuclear envelope protein complex, LC3-networking protein, which degraded during autophagy. Its expression is negatively correlated with proteins distraction during autophagy, accordingly deposition of p62 is concomitant with autophagy failure [61, 62]. The FcγR is the Fc receptor in IgG—Fc receptors are pro-inflammatory proteins located on certain cells’ surface—which involved in the inflammatory phase of nephropathy incidence and progress [63, 64]. FcγR mediated the progress of inflammation and contributed in CKD [65] via supporting the assembly of interleukin 12 in the dendritic cells, carrying out manufacture of INF-γ and other inflammatory markers [66], leading to NF-κB downstream activation. FcγR can also activate LC3 recruitment to phagosomes containing IgG, demanding development of ROS by NADPH oxidase [67]. It participates in glomerular damage [68]. Reliable with this evidence, the outcomes of this study showed a downregulation of the mRNA gene expression ratios of Beclin 1, LC3, along with upregulation of FcγR, p62 gene expression ratios in kidney tissues of R, TAA, and R/TAA-intoxicated animals. However, SP administration upregulated the gene expression ratios of renal Beclin 1 and LC3, whereas downregulated FcγR and p62 gene expression in kidney tissues of R, TAA, and R/TAA-intoxicated animals. Accordingly, SP can maintain autophagy.

AMPK is a central controller protein that, during normal physiology and pathophysiological CKD, performs kidney significant functions [69]. It is a predominant cellular energy homeostasis modulator [70]. It is additionally intensely coupled with both inflammation and autophagy [69, 71]. AMPK protein expression ratio was upregulated during hypoxia. Hypoxia leads to increases in ROS production, a decline of oxygen supply, and failure of oxidative phosphorylation in the mitochondria, causing an enhancement of AMP/ATP ratio. Enhancement of AMP/ATP ratio induces liver kinase B1 to activate AMPK pathway. Hypoxia also leads to an increase of Ca²⁺ production, activating Ca²⁺/calmodulin-dependent protein kinase 2 (CaMKK 2). Subsequently, CaMKK2 induces the phosphorylation and activation of AMPK. On the other hand, ROS deactivates HIF-hydroxylase enzymes, thus enhancing the release of HIFs; this subsequently upregulates AMPK activity [72, 73]. The AMPK and p-AMPK can activate autophagy by repressing mTOR and upregulating p62 [74–76]. The mTOR pathway is a central modifier in the autophagy development. Phosphorylation of AMPK keeps up autophagy kinase, which generally prompts mitochondrial interceded autophagy [77]. Actuated AMPK can hold back mTORC1, which opposing autophagy [77]. The current data demonstrated increases in HIF-1α and Ca²⁺ levels accompanied by upregulation of p-AMPK and p-mTOR protein expression ratio in kidney tissues of R, TAA, and R/TAA-intoxicated animals, whereas SP administration declined HIF-1α and Ca²⁺ levels and upregulated p-AMPK phosphorylation and consequently downregulated p-mTOR ratio.

Collectively, autophagy, apoptosis, and inflammation are effectively correlated. The apoptotic proteins can modulate autophagy, and some autophagic proteins can trigger apoptosis. Caspases play fundamental functions in controlling apoptosis and autophagy. Caspases that activated during apoptosis can cleave and inactivate Beclin-1 [71]. On the other hand, the pro-inflammatory cytokines TNF-α, IL-1B, IL-6, IL-17, and IFN-γ can encourage autophagy. However, autophagy can modulate the production of these cytokines [78]. On the other hand, the data proved that SP administration downregulated the AMPK protein expression ratio in kidney tissues of R, TAA, and R/TAA-intoxicated animals.

MicroRNAs, which are tiny noncoding RNAs, involved in normal physiology and pathogenesis of chronic kidney disorders. They bind to mRNAs, inhibiting target genes expression [5, 79]. MiR-1 participates in oxidative stress-related cardiovascular diseases. MiR-1-overexpression in the cardiomyocytes observed to induce high levels of ROS and may suppress the posttranscription of some antioxidant enzymes, including SOD1 [80]. Moreover, miR-1 expression was upregulated due to H₂O₂ treatment-induced apoptosis, mitochondrial impairment, and release of Cyc-C in pluripotent stem cells [81]. In addition, in an in vitro study, blocking the miR-1 gene expression prevents myocardial fibrosis, which may correlate with regulation of p-AMPK and LC3B expressions, thus restoring the autophagy instability [82]. Correspondingly, miRNA-146a is involved in the progress of CKD. Downregulation of the miRNA-146a gene expression is associated with renal inflammation, via activation of IL-1β–TNF-α–NF-κB signaling [79]. The miRNA-1 gene expression ratio was increased, whereas the miRNA-146a gene expression was downregulated in the kidney tissues of R, TAA, and R/TAA-intoxicated animals. These results suggested the involvement of miR-1 and miR-146a in the kidney injury. Accordingly, miR-1 may associate with the production of ROS, inhibition of the antioxidant enzymes, induction of apoptosis, and autophagy in kidneys of intoxicated animals. However, miR-146a is responsible for the inflammation progress of kidney during chronic toxicity of R, TAA, and R/TAA. SP administration regulated gene expression ratio of miR-1 and miR-146a in kidney tissues of different intoxicated groups. SP contains numerous bioactive compounds, including C-phycocyanin, vitamins, and minerals, which encourages its nephroprotective activity [18, 27, 28].

Spirulina is rich source of antioxidant phytochemicals, such as β-carotene, phycocyanin, tocopherols, essential amino acids, phenolic compounds, microelements (K, Na, Ca, Mg, Fe, Zn), and polyunsaturated fatty acids, such as γ-linolenic acid, which convince antioxidant–anti-inflammatory properties, regulation of macro and microelements, and after all nephroprotective impact [18–22, 27, 28, 83, 84].

Conclusion

This study highlighted the nephroprotective effect of SP extract on R, TAA, and their combined R/TAA toxicities in rats. This apparent protective effect of SP is mediated by regulation of the miR-1 and miR-146a gene expression, preventing release of ROS, inflammation, apoptosis, and autophagy via monitoring AMPK/mTOR route. Subsequently, administration of SP could be a convenient food supplement for protection against R and/or TAA-induced nephrotoxicity.

Authors’ Contribution

A.A.S.: Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, and reviewing.

A.F.M.I.: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review and editing.

Acknowledgments

The authors are grateful to Prof. Dr. Ahmed Osman, Professor of Histopathology, Department of Pathology, Faculty of Veterinary Medicine, Cairo University for his assistance in setting up the histopathological study.

Conflicts of interest

None declared.